124 Chapter 4 Figure 3. Supervised analysis of blood granulocyte and monocyte subsets after severe burn injury. Flow cytometry results of: (A) Neutrophils (CD15+CD16+ granulocytes). (B) Eosinophils (CD15+CD16¯CD9+ granulocytes). (C) Immature neutrophils (CD10dim neutrophils). (D) Mature neutrophils (CD10bright neutrophils). (E) Classical monocytes (CD14brightCD16¯ monocytes). (F) Intermediate monocytes (CD14brightCD16+ monocytes). (G) Non-classical monocytes (CD14dimCD16+ monocytes). Number of subjects per time interval is shown on top of the graphs. Values of burn wound patients and healthy controls (HC) are shown as mean (line and dots) ± standard deviation (colored band). Asterisks indicate significant differences in time within the burn patient group (linear mixed model analysis): *p < 0.05; **p < 0.01. Significant differences of outcomes in burn patients on PBD 0-3 compared to healthy controls are indicated by × (×××p < 0.001). Burn Injury Induces an Increase in CCR4 and CCR6 Expressing CD4+ T Cells and Tregs From the Second Week After Injury Onward Although burn injury did not significantly alter the total number of lymphocytes, unsupervised analysis of the lymphocyte flow cytometry panel revealed changes in the T cell composition (Figure 4). Four main clusters of lymphocytes could be discriminated: CD4+ T cells (nodes 1–7), Tregs (nodes 6, 7), CD4¯ T cells (nodes 8–12) and CD3¯ lymphocytes (nodes 13–16) (containing B cells and NK cells) (Figure 4A). In the CD4+ T cell cluster, the CCR4¯CCR6¯ T cells (node 4), among which could be naïve T cells, decreased upon burn injury. CCR4+CCR6+ and CCR4¯CCR6+ T cells (nodes 1 and 2, respectively) increased in week 2 and remained elevated in week 3 and 4. Two regulatory T cell populations were distinguished: CCR4+CCR6¯ and CCR4+CCR6+ Tregs (nodes 6 and
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